Drinks Like a Fish – Student Handout #3
Are the Toxic Effects of Alcohol Due to Oxidation?
Instructions for dilutions of ethanol and nicotinamide.
1. Make a stock solution of ethanol (10%)
Depending on the concentration of ethanol you have on hand (e.g., 100%, 95%, or 70%),
make a 10% stock solution in egg water. Make about 10 mls. Use the table below to
calculate how much of each reagent to use. The first line is filled in for you if you are
starting with 100% ethanol.
Starting ethanol
concentration
# of mls of
ethanol
# of mls of total # of
egg water
mls
100%
1 ml
9 mls
95%
1 ml
70%
1 ml
10 mls
2. Make a stock solution of the antioxidants nicotinamide (500 mM).
First determine the molecular weight (MW) of the compound, the proposed concentration
for treatment, and whether the compound is soluble in an aqueous (i.e., water) or
organic solvent (i.e., DMSO).
Nicotinamide MW: ________________________
Nicotinamide Solubility: ____________________
How many grams of nicotinamide do you need to make a 500 mM solution in 10 mls?
Your teacher will weigh out the appropriate amount and dissolve in 10 ml water.
3. Make working solutions.
0.2 % ethanol (in egg water)
Stock ethanol
concentration
Working ethanol
# of mls of
concentration
stock ethanol
10%
0.2%
# of mls of
egg water
total # of
mls
100 mls
5 mM nicotinamide (Nic) (in egg water)
Stock Nic
concentration
Working Nic
concentration
500 mM
5 mM
# of mls of
stock Nic
# of mls of total # of
egg water
mls
100 mls
Drinks Like a Fish – Student Handout #3
5 mM nicotinamide (Nic) (in 0.2% ethanol)
Stock Nic
concentration
Working Nic
concentration
500 mM
5 mM
# of mls of
stock Nic
# of mls of
0.2% ethanol
total # of
mls
100 mls
It’s best to prewarm the solutions to 27º-28º C if you are doing the entire experiment at that
temperature. If not, just use room temperature.
Set-up your treatment plates (6-well plates)
1. Take a clean 6-well plate, labeling the lid with code letters according to the chart below
(A-F). You have 4 treatment conditions: no ethanol (control), 0.2% ethanol, 5 mM
nicotinamide, and 5 mM nicotinamide in 0.2% ethanol. There are enough treatment wells
to do duplicate the two ethanol-exposed conditions. Doing duplicate conditions
increases your accuracy, and is helpful in case you make a mistake.
Well Label
A
B
C
D
E
F
Solution for Experimental Treatment
Control – Egg water alone
0.2% Ethanol (made in egg water)
5 mM Nicotinamide in egg water
5 mM Nicotinamide in 0.2% ethanol
Duplicate of Well B
Duplicate of Well D
2. You can label the wells as follows (use a “Sharpie”)
Label the net-inserts the same way.
A
B
C
D
E
F
Drinks Like a Fish – Student Handout #3
3. Add 2 mls of the corresponding solutions (pre-warmed) to each well (use your table
above)
4. Add the net-inserts into the wells
5. Add the remaining 7 mls of solution into the net well-insert (this helps avoid air
bubbles)
Expose the 4 day old zebrafish to the treatment solutions
1. Transfer 10 zebrafish into the net well-inserts containing 9 mls of prewarmed egg water
(with or without alcohol or nicotinamide) using a pipette
2. Place the lid on the plate and then wrap the plate with parafilm or plastic wrap.
3. Put plates in incubator/oven at 27º-28º C for 18-24 hours. If you incubate at room
temperature, it will take longer to produce the neuromast damage.
4. The next day, fill 3 more 6-well plates with fresh egg water at 27º-28º C (9 mls/well). Be
sure the net wells themselves are labeled and not just the 6-well plate!! Transfer the net
wells (individually) into one of the new plates containing fresh egg water. Allow them to
sit for 5 minutes. Repeat two more times. This is a “wash” step and ensures that no
residual ethanol remains.
5. Your fish are now ready to be tested in the super-duper acoustic startle apparatus. Go
to Student Handout #2.
Attention!!
Before doing any testing, you need to be “blinded” by the treatment well. Your teacher will
reassign the well designations with new letters and keep the “code” secret until your experiment
is over. This will avoid any bias on your part while you are giving your fish their movement
scores. After the experiment is over, your teacher will “break the code” and you can write the
actual treatment into your data column.